Sex-determining region Y-box 2 (SOX-2) has been considered a biomarker of lung cancer. SOX-2 is a transcription factor key to maintaining stem cells, essential for early development, that is expressed in adult lung tissue (Lu et al., 2010). SOX-2 overexpression has been found in all types of lung cancer (SCLC and NSCLC). While a cut-off for its clinical application has not been standardized, it is a good biomarker candidate and also a potential therapeutic target (Karachaliou et al., 2013). Aydin and Sezgintürk (2017) reported the electrochemical detection of SOX-2 in serum samples with a novel immunosensor via the modification of a disposable indium tin oxide (ITO) thin film-based electrode with carboxyethylsilanetriol (CTES). Later, Regiart et al. (2020) proposed a microfluidic electrochemical immunosensor for SOX-2, specifically, a modified electrode with gold nanoporous structures (NPAu) that uses the dynamic hydrogen bubble template (DHBT) method.

Carcinoembryonic antigen (CEA) is a glycoprotein that is normally produced in very low levels by some cells in the body, but its levels can rise in the blood of individuals with certain types of cancer, including lung cancer, where it has a standardized cut-off of 3.2 ng/mL (Okamura et al., 2013). CEA is one of the most used electrochemical biomarkers, although its use as a single biomarker for disease diagnosis and prognosis is still controversial (Grunnet and Sorensen, 2012). As a first example, Paimard et al., 2020 presented an electrochemical immunosensor based on carbon paste electrodes (CPE) modified with honey nanofibers (HNF) by electrospinning, which in turn were decorated with gold nanoparticles (GNPs) by electrodeposition and multi-wall carbon nanotubes (MWCNTs) functionalized with carboxylic acid groups. Then, Zhou et al. (2022) described an electrochemical biosensor based on dual-signal amplification through electrically mediated atom transfer radical polymerization (eATRP) to significantly improve the level of detection (LOD). The subsequent covalent conjugation of 2-bromo-2-methylpropionic acid (BMP) allowed eATRP initiation to graft numerous ferrocene methyl methacrylate (FMMA) monomers onto the modified electrode under electrocatalysis. A rather interesting design by Wang X. et al. (2022) describes a “dual-signal off” electrochemical immunosensor based on the combination of two functionalized solid supports. On one hand, Prussian blue nanoparticles (PBNPs) covered with gold nanoparticles-reduced graphene oxide (Au-rGO), and, on the other hand, mesoporous silica nanoshells coated with copper peroxide (CuO₂@SiO₂) nanocomposites allow the release of Cu²⁺ and H₂O₂ under acidic conditions. The generated Cu²⁺ replaces the high-spin iron (Fe^III) in the PBNPs, which in turn reduces the PBNPs’ oxidation peak current.

Although an accurate determination of CEA may be used as a biomarker in a clinical setup, the accuracy of cancer diagnosis can be increased by detecting several biomarkers in a given sample. Our search rendered several assays developed for multiple and simultaneous biomarker determination in addition to CEA. Ning et al. (2018) developed an immune sandwich structure with antibodies for CEA, alpha-fetoprotein (AFP), neuron-specific enolase (NSE), and cytokeratin-19-fragment (CYFRA21-1) on a microarray coupled with scanning electrochemical microscopy (SECM). Upon addition of sample proteins and HRP-labeled antibodies, HRP catalyzed the oxidation of hydroquinone (H₂Q) to form benzoquinone (BQ) in the presence of H₂O₂ on the spots. To avoid the need for cleanroom fabrication, Wang et al. (2019) developed a paper-based electrochemical aptasensor for the detection of carcinoembryonic antigen (CEA) and NSE. To achieve the simultaneous detection of the two markers, the authors used two microfluidic channels and two screen-printed carbon ink working electrodes. The electrodes were modified with amino-functional graphene-thionin-gold nanoparticles (NG-THI-AuNPs) and Prussian blue-poly(3,4-ethylenedioxythiophene)-AuNPs (PB-PEDOT-AuNPs) nanocomposites. A different approach to detecting two biomarkers was used by Li et al. (2020), who proposed a novel electrical signal difference strategy for CEA and AFP detection employing a glassy carbon electrode (GCE). Recently, Zhang R. et al. (2022) broadened the type of sample that could be analyzed by developing a portable multi-channel interdigitated organic electrochemical transistor (SiOECTs) device for noninvasive CEA, NSE, and cancer antigen 125 (CA125) in sputum. Later, simultaneous electrochemical immunodetection of CEA and CA125 was reported by Liu et al. (2022), who developed an immunosensor based on copper-tetrakis (4-carboxyphenyl) porphyrin (Cu-TCPP) nanomaterials and toluidine blue (TB) plus PB as the indicators with a different redox signal. As a last example, Fan et al. (2022) approached the multiplexing detection using a tri-channel electrochemical immunobiosensor for CEA, NSE, and CYFRA21-1 detection in exosomes by using indium tin oxide electrode (ITO) functionalized with (3-aminopropyl) triethoxysilane (APTES) (Figure 4).

Several cell-surface glycoproteins that are overexpressed in lung cancer have been used as biomarkers in cancer diagnosis, and their applicability in serum analysis needs better understanding.

For CD44, a transmembrane glycoprotein that is widely expressed on the surface of cells in the majority of normal and carcinomatous tissues, Białobrzeska et al. (2021) used hyaluronic acid (HA) as a sensing element to design a label-free electrochemical sensor for CD44 based on ligand–protein interactions. A representative approach in the liquid biopsy field is the work by Zhang J. et al. (2021), who reported a micropatterned electrochemical aptasensor integrated with a hybridization chain reaction (HCR) signal amplification method using anti-CD63 aptamer-functionalized gold microelectrodes for EVs capture and anti-epithelial cell adhesion molecule (EpCAM) aptamers for detection. CD63 is a member of the tetraspanin family and a generalized biomarker of total EVs. For its part, EpCAM is a molecular adhesion protein overexpressed in all epithelial-origin cancers, including lung cancer; its combined analysis results in an assessment of circulating epithelial EVs. You et al. (2022) constructed an electrochemical biosensor based on Ti₂CT_x MXene nanosheets decorated with a gold nanoarray for exosome detection using anti-EpCAM aptamers as sensing elements.

Another protein employed as a biomarker is mucin 1 (MUC1), a glycoprotein member of the mucin family that has been found to be increased in lung cancer; its anti-adhesion characteristics and cell signaling implications support the role of MUC1 in the development of human lung cancer and its potential as a biomarker (Saltos et al., 2018). The electrochemical detection of MUC1 was reported by Zhao et al. (2021), who developed a novel hybrid peroxidase mimetic structure to amplify the sensitivity towards the decomposition of H₂O₂ by anchoring Cu₇S₄ and AuNPs on a porphyrin-based porous organic polymer (PPOP) and catalytic hairpin assembly (CHA) to amplify signals. Xie et al. (2022) proposed an electrochemical ratiometric aptasensor with intrinsic self-calibration properties. Co-metal-organic frameworks (MOFs) were employed as signal substances. The gold nanoparticles@black phosphorus (BP) comprising the electrode substrate was modified via an Au-S bond with DNA nano-tetrahedrons (DTN) containing MUC1 aptamers.

Overexpressed growth factor receptors are associated with cancer development and have been studied as biomarkers. Wang et al. (2020) designed an electrochemical origami paper-based aptasensor for label-free detection of epidermal growth factor receptor (EGFR) by employing anti-EGFR aptamers as the bio-recognition element. The hydrophobic areas were created by wax printing, and the electrodes were screen printed. Amino-functionalized graphene/thinine/gold nanoparticle nanocomposites were used to modify the carbon ink working electrode and accommodate the anti-EGFR aptamers through Au-S bonds (Figure 5). Rejeeth et al. (2022) used poly (acrylic acid) (PAA) as an affinity bait molecule to capture and detect the platelet-derived growth factor receptor (PDGFR).

Sumithra et al. (2020) fabricated an impedimetric biosensor for the splicing factor called Src-associated protein in mitosis 68 (Sam68) detection. Sam68 is overexpressed as lung cancer advances to metastatic stages and thus can be considered a biomarker of disease progression. In this case, the affinity capture molecule is an anti-Sam68 antibody, which is immobilized via glutaraldehyde over a GCE modified with polyaniline (PANI). Chen et al. (2020) detected the early LC biomarker neutrophil-activating peptide 2 (NAP2) with two variants of a signal-off/on electrochemiluminescence (ECL) deoxyribosensors. Another example of immunoaffinity capture and detection is the electrochemical immunosensor designed by Kuntamung et al. (2021) for GM2 activator protein (GM2AP) detection. GM2AP is a novel urinary biomarker in LC patients, who can show over a five-fold increase in this biomarker compared to healthy controls. In brief, screen-printed carbon electrodes were modified with polyethyleneimine-coated gold nanoparticles (PEI-AuNP), phosphomolybdic acid (PMA), and anti-GM2AP antibodies. GM2AP detection is based on a decrease in the differential pulse voltammetry (DPV) current response of PMA redox probes proportional to the GM2AP concentrations. Finally, following the immunoaffinity capture strategy, Deepa et al. (2022) reported an amperometric immunosensor to detect p53, a tumor suppressor gene known as the “guardian of the genome” because its principal function is the control of repairing DNA that is highly activated in cancer. This rather simple device was fabricated by immobilizing anti-p53 antibodies onto a pencil graphite electrode (PGE) via adsorption. Table 2 summarizes the principal characteristics of reviewed articles in total LC.

CYFRA21-1 has been extensively evaluated as an NSCLC biomarker and, in consequence, many sensors have been developed for its analysis. It is part of the cytokeratins family and forms the cytoskeleton filaments of all epithelial cells. Its application in LC diagnosis is very well documented, with a diagnosis cut-off of 3.5 ng/mL (Okamura et al., 2013). In 2018, Zeng et al. (2018) reported a sandwich-type electrochemical immunosensor showcasing a composite structure consisting of three-dimensional graphene (3D-G), chitosan (CS), and glutaraldehyde (GA) on a GCE to expand the surface area with exceptional conductivity. Then, in another interesting approach, Meng et al. (2019) presented an electrochemiluminescent (ECL) immunoassay by using molybdenum oxide quantum dots (MoOx QDs). Later, Feng et al. (2021) presented a microfluidic system that combined a microelectrode and a cathodic photoelectrochemical (PEC) biosensor. The amplification mechanism hinges on the p–n junction of AgI/Bi₂Ga₄O₉, with dissolved O₂ serving as an electron acceptor, resulting in the production of superoxide anion radicals (˙O₂−) as the working microelectrode. To further enhance the detection capability, a novel superoxide-dismutase-loaded honeycomb manganese oxide nanostructure (SOD@hMnO₂) was employed as a co-catalyst signal amplification label.

Yola et al. (2021) developed a sensor that employs a composite material comprised of silicon nitride (Si₃N₄) and molybdenum disulfide (MoS₂) incorporated onto multi-walled carbon nanotubes (MWCNTs) as the reaction platform. Additionally, it utilizes core-shell magnetic mesoporous silica nanoparticles@gold nanoparticles (MMSNs@AuNPs) as a signal amplifier. In a study by Yang et al. (2021) that aimed to detect CYFRA21-1, they distributed GNPs on the surface of three-dimensional graphene (3D-G) attached to a GCE with thionine (thi) and m-cresol purple (MCP) as signal tags. In the same year, Wang et al. (2021) presented an electrochemical immunosensor where graphene oxide (GO) was immobilized into the GCE as an immune reaction support. Then, a secondary antibody was conjugated with 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPAD) to be introduced into the sandwich reaction. Finally, the immunocomplex was linked to numerous monomers through reversible addition-fragmentation chain transfer (RAFT) polymerization. In another work, Lu et al. (2021) proposed a novel electrochemical immunosensor based on the ring-opening polymerization (ROP) signal amplification strategy. At the same time, Cheng and Hu. (2021) developed a sandwich-type electrochemical immunosensor based on a combination of gold nanoparticle (AuNPs) decorated Ti₃C2Tx-MXene (Au-Ti₃C2Tx) over a GCE. Another novel sensor proposed by Zhang J. et al. (2021) employed nanoparticle impact electrochemistry (NIE) using silver nanoparticles (AgNPs) as labels. The approach is grounded in measuring the impact frequency and the charge intensity resulting from the electrochemical oxidation of individual AgNPs. Recently, Gu et al. (2022) chose mesoporous carbon CMK-3 to mix with carboxylated multi-walled carbon nanotubes (CMWCNTs) to enhance electron transfer efficiency. Furthermore, gold nanoparticles (AuNPs) were incorporated to offer a multitude of binding sites for antibodies.

In another interesting approach, Feng et al. (2022) developed an immunosensor by encapsulating an electroactive dye, such as methylene blue (MB), to enhance signal amplification. In the same year, Hu et al. (2022) synthesized AuNPs@MoS₂@Ti₃C₂Tx composites, where titanium carbide (Ti₃C₂Tx) was modified with gold nanoparticles (AuNPs) and molybdenum disulfide (MoS₂) and used as a solid support. Finally, Jafari-Kashi et al. (2022) designed a novel label-free electrochemical DNA biosensor based on the oxidation signal of guanine.

In recent years, an increasing number of promising biomarkers with clinical relevance in NSLC have been discovered, and several electrochemical analysis developments have resulted. In this sense, Roberts et al. (2019) developed an electrochemical immunosensor for the swift identification of urokinase-type plasminogen activator receptor (uPAR), an important factor of the fibrinolytic system in tumor progression and metastasis. This investigation employed a cutting-edge ultrasensitive electrode crafted from graphene nanosheets (GNS) functionalized with a monoclonal antibody. Next, Białobrzeska et al. (2021) fabricated a biosensor based on a screen-printed gold electrode functionalized with monoclonal antibodies for impedance quantification of anterior gradient-2 protein (AGR2) that is a protein widely secreted in precancerous lesions, primary tumors, and metastatic tumors (Figure 7). Saputra et al. (2022) developed an amperometric biosensor for the sensitive detection of granzyme B (GzmB) using a dual monomer-based bioconjugate; GzmB is a serine protease highly expressed in immune cells and tumor microenvironments that is a promising biomarker when it is detected in serum. Furthermore, Chai et al. (2022) developed an electrochemical immunosensor for the ultrasensitive detection of pro-surfactant protein B (pro-SFTPB). This proprotein is produced by alveolar cells and released into the alveolar compartments. This sensor utilized innovative binary PdCu mesoporous metal nanoparticles as signal labels and employed black phosphorus (BP) nanosheets and AuNPs for electrode modification. Finally, Taheri et al. (2022) developed a highly effective sensing layer using a dual-template molecularly imprinted polymer (DMIP) for the individual detection of two lung cancer biomarkers: CEA and AFP.

Interestingly, three manuscripts focused on the quantification of specific exosomes with clinical relevance in NSCLC. Wang M. et al. (2022) developed a highly sensitive biosensor capable of detecting programmed dead-ligand 1 positive (PD-L1+) exosomes by utilizing covalent organic framework nanosheets (2D COF NSs) and CRISPR-Cas12a for signal amplification. Then, Sha et al. (2022) presented a DNA-fueled electrochemical sensor for the identification of PD-L1+ exosomes. These exosomes were enriched by using magnetic beads decorated with PD-L1 antibodies and could interact with cholesterol-modified hairpin templates. Then, programmable DNA synthesis starting from the hairpin template triggers a primer exchange reaction and generates a large number of extension products to activate the trans-cleavage activity of CRISPR-Cas12a to enhance the reaction. Finally, Zhang Y. et al. (2022) developed an MOF-based electrochemical liquid biopsy (ELB) platform that offers efficient, sensitive, and multiplexed detection of cancer exosomes by detecting CD9, CD63, and CD81, which are the members of the tetraspanin family employed as an EV-specific marker. Table 3 summarizes the principal characteristics of reviewed articles about NSCLC.